(by pjie2 from cam.ac.uk)
Fri Sep 24 14:28:19 EST 2010
The protein I'm working on doesn't want to express in any culture or in
vitro system I've tried. So far I've been through:
pEXT5-CT vector (T7 promoter, C-terminal His tag). No soluble or
insoluble expression in BL21DE3 cells with autoinduction or with IPTG
induction. No soluble or insoluble expression using coupled T7
transcription/translation systems, either with E. coli extract or
pT7CFE1 vector (T7 promoter, C terminal His tag, ribosome entry site and
3'UTR optimised for use in vitro transcription/translation with
mammalian extracts). No soluble or insoluble expression with
reticulocyte lysate transcription/translation system.
pGEX(KG) vector (tac promoter, N terminal GST fusion). No soluble or
insoluble expression using autoinduction or IPTG induction.
pEGFP-N1 vector (CMV promoter, N terminal GFP fusion). No soluble or
insoluble expression in two different mammalian cell lines.
All four of the above systems have worked fine in our hands with control
genes (and some other genes I'm working on), so it's nothing wrong with
the kits/cells or our lab technique. For the latest attempt at in vitro
transcription/translation, we isolated the mRNA from the reaction and
sequenced it, so we know the constructs are being expressed and is
in-frame etc. The protein just doesn't want to translate, no matter what
system we use.
I guess my protein of interest just really doesn't like being expressed
in culture! Maybe it needs particular cofactors / chaperones in order
to be translated? Does anyone have any suggestions what to try next?
The protein itself is about 50kDa, specific to round spermatids in the
testis. Spermatids are pretty weird cells, so it's certainly possible
that my protein isn't happy in any other cell type. Unfortunately round
spermatids don't grow in culture, so transfecting spermatids directly
isn't an option.
About all I can think to try is to add some testis extract to an in
vitro transcription/translation reaction and hope it supplies whatever
the missing factors are. Has anyone tried anything like that before,
and is it a remotely sensible thing to try? If so, what's the best way
to go about it - homogenise testis tissue in RIPA buffer or PBS and just
add some to the reaction?
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